The present invention relates to the discovery of a new bacterium which has been found to attract oyster larvae by the production of compounds involved in metabolism and melanin synthesis. More specifically, the present invention contemplates a method for inducing the settlement and metamorphosis of Crassostrea virginica larvae by induction with certain metabolic substances produced by the present bacterium and its mutagenically altered variants. Furthermore, the present invention is directed to other and derivative metabolic products which can be employed for their desired utility and application.
The formation of pioneer microbial communities on submerged surfaces appears to be beneficial to subsequent attachment and development of many invertebrate larvae. A number of investigations have established a general pattern of periphytic succession for colonization of clean surfaces immersed in seawater. In the initial phase after possible coating by organic matter, bacteria attach to such a surface and begin to grow, forming microcolonies within several hours. Subsequently, diatoms, fungi, protozoans, micro-algae and other microorganisms attach to the surface, forming what has been termed the primary slime layer. This primary microbial colonization appears to be a prerequisite for the final stage of succession in which microorganisms, viz., invertebrates, attach and grow on the surface. Although most surfaces are eventually colonized, the rapidity and extent of the process eventually colonized, the rapidity and extent of the process depends on the nature of the surface material, the prevailing environmental conditions and the composition of the periphytic populations.
Two invertebrate species for which some information has been ascertained concerning the effect of the periphytic organisms on their induced metamorphosis are the sea urchin, Lytechinus picturs, and the hydroid, Hydractinina echinate. It has been determined that for Lytechinus, the responsible factor is a low molecular weight bacterial by-product, probably proteinaceous having a molecular weight less than 5000 daltons. It has also been found that planulae larvae of Hydractinia metamorphose in response to a product emitted by certain marine, gram-negative bacteria at the end of their exponential growth phase. If these bacterial cultures are subjected to osmotic shock, the activity shows up in the supernatant, suggesting that the critical product is a soluble factor rather than a bound one. When Hydractinia are kept in sterile conditions, they do not metamorphose.
In a series of experiments designed to determine the physiological mechanism by which the stimulus activates metamorphosis, it has been demonstrated that the inducer may operate by stimulating the Na.sup.+ /K-ATPase of larval cell membranes. Such findings are the first real steps toward understanding how larvae can mount a broad spectrum morphogenetic response to specific environmental stimulation. Moreover, recent reports have shown that Vibrio sp. excretes a product that induces metamorphosis of the chidarian, Cassiopea andromeda. Other investigations demonstrate that larvae of the marine annelid, Janua brasiliensis, settle on certain microbial films and that certain specific bacteria may induce metamorphosis. These observations suggest that the processes are mediated by larval lectins binding to extracellular polysaccharides or glycoproteins produced by the bacteria.
In both the natural environment and in oyster mariculture operations, the setting process, whereby planktonic oyster larvae alight on an oyster shell or plastic sheet and undergo metamorphosis to form attached oyster spat, is crucial to successful oyster development. It is also known that the larvae of Ostrea edulis, the European oyster, prefer setting on surfaces covered with a film of bacteria and diatoms. Natural periphytic microbial populations are, therefore, significant in successful oyster setting. The same situation is likely to be true of oyster mariculture, since a rich source of bacterial flora has been associated with oyster larvae and larval food sources in hatcheries. In some cases, bacteria have also been implicated in the death of oyster larvae. Since the presence of microorganisms significantly affects oyster development, improved knowledge of the biology of these microorganisms and particularly an understanding of their beneficial and/or deleterious effects on developing oysters, will further improve oyster setting and development in both natural and artificial settings.
Oyster larvae display three characteristic patterns toward organic compounds and microorganisms, i.e., positive, inactive and negative chemotaxis. In one particular study, a marine pseudomonad was attractive to larvae while a marine yeast elicited no response. It has also been suggested that an alga, Isochrysis, may produce extracellular oyster attractant. Conversely, it is known that oyster larvae do not set preferentially on surfaces to which a marine isolate, Hyphomonas neptunium, is affixed. It is believed that H. neptunium does not antagonize settlement, but rather that it competitively establishes itself on surfaces and excludes bacterial species which would be beneficial to oyster settlement.
The question, however, of which of the periphytic microorganisms and which of their products specifically attract or promote the setting and subsequent development of oyster larvae has not been answered heretofore. Free swimming larvae, shortly after spawning, seek a suitable place to settle and attach themselves. A number of environmental conditions are involved in settlement, salinity and nutritional availability are probably the most important. But once larvae are satisfied with these initial conditions, they appear to respond to a biochemical cue to settle and attach themselves. That biochemical cue is released by a pigmented bacterium which adheres strongly to surfaces such as oyster shells and which is the subject of this invention.